重组ACTH(4-10)与GDNF融合蛋白及其生物活性研究

通过PCR方法构建了促肾上腺皮质激素4-10 (ACTH(4-10))与胶质细胞源性神经营养因子（GDNF）的融合基因,并将它重组克隆到表达载体pET-28a(+)中,构建表达质粒pET-ACTH(4-10)-GDNF,转化大肠杆菌BL21(DE3),经IPTG诱导可高效表达ACTH(4-10)-GDNF融合蛋白.用Ni²⁺-NTA树脂一步法纯化目的蛋白,纯度达85％以上.纯化和复性的ACTH(4-10)-GDNF融合蛋白能显著促进脊髓神经元存活,作用强于ACTH(4-10)及GDNF蛋白.     

醋酸铅对体外培养大鼠肾小管上皮细胞的毒性损伤

探讨醋酸铅对体外培养的大鼠肾小管上皮细胞系HBZY-1的细胞毒性效应.用不同浓度的醋酸铅(30、40、50 μmol/L)作用HBZY-1细胞24 h、48 h和72 h,采用CellTiter-Glo(R)萤光细胞活性检测盒测定细胞存活情况;用流式细胞仪检测细胞凋亡率.结果显示经30、40、50μmol/L醋酸铅处理...     

骨桥蛋白RGD黏附序列多拷贝短肽的表达、纯化及生物学活性测定

目的:用大肠杆菌表达骨桥蛋白RGD黏附序列6拷贝短肽,经分离纯化后检测其生物学活性.方法:运用基因重组技术,将骨桥蛋白RGD黏附序列的核酸片段首尾相连,与携带GST编码序列的原核表达载体连接构建融合蛋白表达质粒pGEX-3X-RGD.将重组质粒转化宿主菌后,对诱导融合蛋白表达的条件进行优化.表达产物GST-RGD经谷胱甘肽-亲和层析纯化后,分别检测其对骨桥蛋白诱导的血管平滑肌细胞黏附和迁移的影响.结果:所构建的含有6个拷贝短肽的GST-RGD融合蛋白可在大肠杆菌中以包含体的形式进行表达.用十二烷基肌氨酸钠变性溶解包含体及透析复性后,经亲和层析可得到高纯度的GST-RGD(6)融合蛋白.GST-RGD(6)融合蛋白能特异性的抑制骨桥蛋白诱导的血管平滑肌细胞的黏附和迁移.结论:骨桥蛋白RGD黏附序列6拷贝短肽可在大肠杆菌中高效表达,纯化的GST-RGD融合蛋白具有抑制血管平滑肌细胞黏附和迁移的活性.     

利用冰核蛋白N-端结构域在大肠杆菌表面展示人存活蛋白

存活蛋白(survivin)是高度特异的广谱肿瘤相关抗原,利用冰核蛋白(ice nucleation protein,INP)表面展示系统研究在大肠杆菌细胞表面展示人存活蛋白的可行性。将人存活蛋白基因片段和报告基因Cherry融合到冰核蛋白N-端,构建表面展示载体p ET-INP-CHY-SUV并转化大肠杆菌BL21(DE3)。重组菌经IPTG诱导后可检测到红荧光,荧光强度在胰蛋白酶的作用下降低,完整细胞ELISA实验及免疫荧光实验可检测和观察到绿荧光,表明人存活蛋白在重组菌中得到表达,并且成功展示于细胞表面,为进一步研制新型的肿瘤DNA疫苗奠定基础。     

动物细胞培养及单克隆抗体

924039抗肺炎球菌英膜多糖4、8、22F和19A门gF型的鼠单克隆抗体〔英」/Kolberg,J.“·了APMIS一1092,100(i)一91~04〔译自DBA,1902,11 (8),92一04640〕 用12声923价的荚膜多糖肺炎球菌疫苗Pneu-movaxN皮下注射BALB/c下小鼠。33周后即融合前4天,动物得到6产g疫苗。或者用6声g疫苗经皮下注射免疫小鼠2次(间隔1周),44周后(融合前4夭),小鼠获得10声9 19F多糖。利用标谁的技术融合脾细胞和NSO骨髓瘤细胞,以23价疫苗为外壳抗原筛选第一次融合获得的杂种细胞上清液。用个别疫苗组分组成的小组进行了抗皿实验之后,选择出3种杂交瘤并克隆之…     

《生物工程讲座》(Ⅳ)——基因工程的理论及应用(三)真核细胞的基因转移技术及其应用

真核细胞基因的转移可以通过转移整个染色体组,也可以转移一组染色体或重组的DNA分子到细胞中去,这些,若从方法学上加以分类有: 1.细胞杂交法; 2.微细胞(microcell)融合; 3.染色体介导的基因转移; 4.DNA介导的基因转移; 5.细胞微量注射法。用细胞杂交方法可以转移整个基因组,而用微细胞转移基因则是用微细胞作供体。在微细胞中有一个或几个染色体与遗传性完整的受体融合,因此,在微细胞系统中只有一个或很少的染色体转移到受体中去。用染色体转移基因是由受体细胞经细胞内吞作用(endocytosis)来     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

牛病毒性腹泻病毒Erns-E2融合蛋白在果蝇S2细胞中的表达与鉴定

目的:利用果蝇S2细胞表达牛病毒性腹泻病毒(BVDV)Erns-E2融合蛋白,并对其抗体结合能力进行鉴定.方法:用RT-PCR方法扩增BVDV NADL株Erns和E2蛋白的编码基因,利用(G4-S)3柔性15肽基因将扩增的2个基因连接,再与昆虫表达载体pMT/BiP/V5-His连接构建重组表达载体pMT/BiP/V5-His-E(MS)-E2,将后者与筛选质粒pCoBlast共转染果蝇S2细胞后表达Erns-E2融合蛋白.并对表达产物进行鉴定.结果:SDS-PAGE结果表明,融合蛋白相对分子质量为76 800;Westem blotting检测表明,该融合蛋白具有与BVDV抗体良好的结合能力.结论:BVDV的Erns-E2融合蛋白能在果蝇S2细胞中进行表达;经鉴定,表达产物具有良好的抗体结合能力,可用于抗原检测.     

雌百灵端脑新生神经前体细胞的产生和分布并与白腰文鸟的比较

用BrdU标记DNA的ABC免疫细胞化学方法,观察雌性蒙古百灵端脑神经前体细胞的产生和分布特点,并与白腰文鸟作比较。结果如下:1.在百灵和白腰文鸟胸肌注射BrdU短时程组(存活1天),在端脑室带区外侧壁(LVZ)有大量的标记细胞,新生神经细胞起源于端脑室带区(VZ)中的增殖细胞层,并在纹状体腹侧的VZ形成标记细胞增殖热点,如在百灵和白腰文鸟靠近中缝线处的外侧纹状体(LSt)与内侧纹状体(MSt)腹侧的LVZ形成标记最多的‘第1增殖热点’区;在靠近中缝线处LVZ的头端形成密集的新生标记细胞,形成‘第2增殖热点’区;在百灵LSt尾端的LVZ标记细胞形成‘第3增殖热点’,但白腰文鸟此脑区的标记细胞较少。2.在百灵胸肌注射BrdU长时程组中5天起,大量的LVZ的标记细胞开始迁移,存活5-30天期间在高级发声中枢(HVc)和高位发声运动中枢-古纹状体栎核(RA)有新生标记细胞,在端脑靠近LVZ的区域有较多的标记细胞。但在雌性白腰文鸟胸肌注射BrdU存活30天期间,在HVC、RA内未见到标记细胞。结果提示雌百灵端脑HVc和RA不断地产生新生神经细胞,这可能与雌性需要不断地感知、识别雄百灵鸣唱的新语句有关,而白腰文鸟不需要这种功能。     

pEGFP-HPV16E7重组融合蛋白质粒的构建及其表达

应用基因重组技术,构建增强绿色荧光蛋白(EGFP)与人乳头瘤病毒16型E7(HPV16E7)的重组融合表达质粒,经限制性内切酶酶切鉴定和PCR分析后,用基因转染技术将其导入小鼠肝癌细胞,荧光显微镜下观察融合蛋白的表达.酶切鉴定和PCR分析证实重组质粒中插入目的基因片段的大小、方向和插入位点均正确,在转染的小鼠肝癌细胞中观察到绿色荧光蛋白的表达.构建的pEGFP-HPV16E7融合表达质粒能直观地反映转染细胞中EGFP-HPV16E7融合蛋白的表达.由于转化率与表达率融为一体,故有利于对转染细胞的筛选,缩短转染细胞在体外的筛选的时间适用于对HPV16E7分子生物学特性、致瘤机理及APC提呈等的研究.为建立表达HPV16E7的实体瘤动物模型奠定了基础.     

Increase in intracisternal A-type particles in Friend cells during inhibition of Friend virus (SFFV) release by interferon or azidothymidine.

Interferon or azidothymidine inhibition of Friend virus (FV-SFFV) release in Friend erythroleukemic cells results in a 10–20-fold increase in intracisternal A-type particle number within 3–4 days of treatment. Inhibition of Friend or Moloney helper virus by interferon in fibroblast producer cells does not result in a similar increase in intracisternal A-type particles. Friend cells with marginal FV production but with high levels of A-type particles do not change their A-type particle levels upon exposure to interferon. These data suggest that A-type particle expression in Friend cells may be linked to the presence of the transforming SFFV. No marked increase of viral RNA levels during inhibition of virus release is observed in Friend cells.     

Comparison of chloride transport in mouse erythrocytes and Friend virus-transformed erythroleukemic cells.

Friend erythroleukemic cells, which grow continuously in tissue culture, resemble in many respects early precursors of mouse erythrocytes. To determine whether or not the membranes of these cells exhibit the rapid and selective exchange of chloride, a specialized feature of the mature erythrocyte membrane, anion fluxes were compared in Friend cells and mouse erythrocytes. The chloride flux in Friend cells at 37 degrees C was about 800-fold lower than in mouse erythrocytes (extrapolated from data at lower temperatures). This difference could not be accounted for by the somewhat lower chloride concentration in Friend cells relative to erythrocytes. Comparison of chloride and sulfate fluxes revealed that the Friend cells had over a 1,000-fold lower selectivity for chloride versus sulphate than did the mouse red cells. The temperature dependence of chloride fluxes in Friend cells corresponded to an Arrhenius activation energy of 17.9 kcal/mol, in contrast to over 30 kcal/mol for mature red cells. The chloride flux in Friend cells was also 10-fold less sensitive to the inhibitor, furosemide, than was the flux in mature red cells. The selective chloride exchange system of the mature erythrocyte therefore does not seem to be functional at the stage represented by the Friend cell, and must appear at some later stage of erythroid maturation.     

Antigen-driven T cell clones can proliferate in vivo, eradicate disseminated leukemia, and provide specific immunologic memory

The aim of the current study was to determine the ability of antigen-driven cloned helper cell independent cytotoxic T lymphocytes (HITc) to proliferate and to survive in vivo and to mediate tumor therapy. The HITc clone utilized (denoted 1.B6) was specifically cytolytic to FBL-3, a syngeneic Friend virus-induced murine leukemia. Activation in vitro (48 hr) with FBL-3 induced secretion of interleukin 2 (IL 2), expression of IL 2 receptors (IL 2R), and in vitro proliferation. These cells could be "rested" for several weeks without stimulation, which resulted in reduced expression of IL 2R; however, restimulation with antigen resulted in reinduction of IL 2R and proliferation. The ability of cloned HITc to proliferate and to survive in vivo was examined in cyclophosphamide (CY) pretreated donor mice congenic for the Thy-1 gene. Adoptively transferred cloned HITc could be found in large numbers, and were widely distributed in vivo 1 wk after transfer. In tumor therapy, 1.B6 cells when injected into a site of tumor (i.p.) and used as an adjunct to CY were effective against disseminated FBL-3. In this circumstance, cloned 1.B6 cells could be recovered from cured mice 125 days after transfer and were shown to specifically lyse tumor and proliferate in vitro in response to FBL-3. Thus as an adjunct to CY, tumor-specific cloned HITc are capable of eradicating disseminated leukemia, persisting long-term in vivo, and providing specific immunologic memory.     

The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.     

Cell surface charge alterations occurring during dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells

Dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells caused a decrease in net negative cell surface charge which began two days after exposure to the polar solvent and continued throughout the maturation process. Neuraminidase treatment caused a marked reduction in mobility of both untreated and dimethylsulfoxide-treated cells suggesting that sialic acid residues are the major anionogenic moieties of the surface membrane of Friend cells. A decrease in the content of total glycosidically bound sialic acid in dimethylsulfoxide-treated cells also occurred. The findings provide evidence to support an association between erythrodifferentiation of Friend cells and net negative surface charge dependent upon sialic acid residues.     

8种致病真菌在5种材料上的存活时间研究

目的了解实验所用常见致病真菌能否在常用医用原材料上生存及生存时间。方法选择8种常见致病真菌,活化后将它们分别接种在5种常用的医用原材料上,接种后及以后的每24h将接种有真菌的医用材料放入巯基乙酸盐液体培养基中并观察能否生存,记录存活时间。结果红色毛癣菌和须癣毛癣菌在棉布、纸板、铁皮、玻璃表面存活25～28d,在地板胶表面存活14～15d;新生隐球菌在棉布、纸板、铁皮表面存活24～27d,在玻璃和地板胶表面存活14～18d;茄病镰刀菌在上述5种材料上存活22～27d;石膏样小孢子菌在棉布、纸板、铁皮和玻璃表面存活20～27d,在地板胶表面存活8d;申克孢子丝菌在棉布、纸板、玻璃和地板胶表面存活17～24d,在铁皮表面存活10d;白念珠菌在棉布、纸板、铁皮和地板胶表面存活15～20d,在玻璃表面存活4d;犬小孢子菌在棉布、纸板、铁皮、玻璃和地板胶上存活时间分别为10d、9d、3d、2d和1d。结论实验所用各种真菌在医用原材料上都能存活,其时间长短不仅与菌种有关,还与所存在的材料有关。真菌在吸水性强、表面粗糙的材料(棉布、纸板)上的平均存活时间长于在吸水性差、表面光滑的材料(玻璃、铁皮和地板胶)。     

Alterations in chloride transport during differentiation of Friend virus-transformed cells.

Friend erythroleukemic cells can be induced by a variety of agents to synthesize hemoglobin and to exhibit other characteristics suggesting erythroid maturation. Upon induction of hemoglobin synthesis with dimethylsulfoxide (DMSO), the chloride flux in Friend cells gradually increases, until after five days of exposure to DMSO (when the hemoglobin content of the cells approaches that of the mature erythrocyte) the flux is three times the value in non-induced cells. A similar flux increase is observed in the presence of a different type of inducer, hypoxanthine, but no increase in flux is seen in the mutant cell line, TG-13, which does not synthesize hemoglobin after DMSO treatment. Thus, the flux increase seems to be associate d with the induction process, rather than being a direct effect of the inducing agent. After DMSO treatment, the sulphate flux decreases and the chloride/sulphate selectivity increases, aswould be expected if the cells were becoming more like red cells. On the other hand, the sensitivity of the chloride flux to the inhibitor, furosemide, and to temperature is the same in the induced as in the non-induced Friend cells, and different from that of the mature red cell. Thus, the anion transport properties of the induced Friend cell are different from those of both the non-induced Friend cell and the mature erythrocyte. Either the system in the induced cell represents an intermediate stage in the development of the mature red cell characteristics, or else the maturation of transport function in the Friend cell differs from that in normal erythrocyte precursors.     

Cell-mediated immunity to friend virus-induced leukemia. IV. In vitro generation of primary and secondary cell-mediated cytotoxic responses.

Primary and secondary cell-mediated cytotoxic responses to FBL-3 cells, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be generated by in vitro techniques as tested by the 125IUdR release assay. The specificity of the cytotoxic reactions appeared to be directed against the Friend type-specific antigen and the FMR (Friend, Moloney, Rauscher) antigen which were also the major antigens for transplantation immunity to FBL-3. In comparison to the primary cytotoxic response, the secondary cytotoxic response was accelerated (detected at an earlier time after sensitization), enhanced (gave much higher levels of cytotoxicity), was also longer lasting, and could be induced by a wide dose range of tumor cells. The secondary response could only be induced with lymphocytes obtained from regressors that were resistant to FBL-3 challenge; lymphocytes from mice with progressive tumor growth had no detectable secondary response. It was found that both induction phase and the effector phase of cytotoxic responses were T cell dependent. The characteristics of these reactions were thus very similar to those obtained with in vivo immunization or challenge, providing a good correlation with in vivo tumor immunity.     

Inhibition of retrovirus-induced disease in mice by camptothecin.

We have previously shown that noncytotoxic doses of camptothecin (CPT), a topoisomerase I-specific antagonist, inhibit retrovirus replication in acutely and chronically infected cells. To evaluate the efficacy of CPT as an antiretroviral drug in vivo, we injected newborn BALB/c mice with Moloney murine leukemia virus and adult NFS mice with Friend spleen focus-forming virus. The Moloney murine leukemia virus-injected mice developed lymphoma, and the Friend spleen focus-forming virus-injected mice developed erythroleukemia. CPT, administrated together with the virus or 1 or 2 days after virus injection, prevented the onset of the disease in both cases. We showed that repeated CPT treatments increased the effectiveness of the drug when administrated 3 days after virus injection. This ability of CPT to inhibit retrovirus-induced disease in vivo without causing any apparent toxic side effects suggests its application as a legitimate remedy for the treatment of retroviral diseases.